Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Abstract

The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex proteinprotein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery. 2-DE historical evolutionGlobal profiling of proteins in healthy versus pathological states is a strategy to discover biomarkers for early diagnostics, disease progression, treatment response and novel targets for therapeutic drugs development. The idea of making an inventory of all the proteins in a cell, tissue or organism with normal or abnormal physiology, was (re)initiated in the middle of the 1990s with the proteome project [1]. Since then, many technologies to make the analysis of the proteome a reality have been united.The perfect technological 'marriage' that made the proteome project feasible was between high resolution 2-DE for separation of large numbers of proteins and MS for identification and characterization of the proteins displayed on this gel [2]. However, before this happy union occured, science and technology devoted to protein study had a long journey until 2-DE and MS combination was possible (Fig. 1). It started in the last century, in 1937, when Tiselius [3] introduced electrophoresis technique as a modification of Reiner's early concept (1927) [4] to analyse a complex mixture of proteins such as serum proteins. Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10]. Only in 1975, was IEF in denaturing conditions, as known today, used to follow the 2-D PAGE and it was described in three independent works [11][12][13]. The most powerful demonstraCorrespondence: Dr. Deborah Penque, Laboratório de Proteó-mica, Departamento de Genética, Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., A...

Signaling Pathways of Proteostasis Network Unraveled by Proteomic Approaches on the Understanding of Misfolded Protein Rescue

“…Proteome profiling of cells/tissues under different conditions (e.g., health vs. disease, treated vs. nontreated) by two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS)-based proteomic approach has contributed to the elucidation of the basic cellular mechanisms of disease by discovering candidate biomarkers and disease targets for new drug development (Penque, 2009).…”

“…In spite of the inability UPR/ER Stress Signalling Pathways Unraveled by Proteomics of 2DE to resolve all proteins present in a sample (e.g., highly hydrophobic proteins, low abundance proteins, extremely basic or acidic pI values, molecular size out of gel limits) or the growing developments in gel-free MS-based approaches, 2DE in combination with MS still remains the most popular technology in proteomics (Penque, 2009).…”

“…There are different 2DE methodologies or possibilities and all require the use of biological and technical replicates to produce meaningful results (Penque, 2009). A guidelines called MIAPE (Minimum Information About Proteomics Experiments) gel electrophoresis that act as checklists of the essential information that should be reported about particular 2DE experimental techniques has been also recently created (http://psidev.sourceforge.net) (see below).…”

“…Metabolic labeling is much more sensitive [<0.1-0.01 ng (Penque, 2009)] than the "blue silver" Coomassie (detection limit 1-100 ng/spot) (Candiano et al, 2004). Moreover, [ 35 S]-methionine labeling detects the presence of a number of proteins that are undetectable by silver staining or Western blotting (Chen and Casadevall, 1999).…”

“…Two specific types of MS analysis can be used for protein identification: the peptide-mass fingerprint (PMF), which involves determination of the masses of the peptides in the digest and is normally performed in a MALDI-TOF instrument, and the amino acid sequencing of the peptides (MS/MS), normally obtained by ESI tandem MS or MALDI-TOF/TOF (Penque, 2009). …”